Due to the introduction of specialized therapies for specific types of cancer, the importance of immunohistochemical staining for diagnosing cancers is increasing. Furthermore, the increasing demand for high throughput in pathology laboratories in combination with the importance of avoiding any procedural errors has emphasized the need for automation of the immunohistochemical procedures.
The majority of tissue obtained from clinical specimens is fixed in a cross-linking fixative such as formaldehyde. This procedure stops the natural degradation of the tissue and thus retains the morphology intact for many years. The fixation forms cross-links between reactive amino acids in the proteins of the tissue in the form of methylene bridges and a poly-oxy-methylene network, which prevents the elution of soluble proteins and retains the molecules in a spatial arrangement that closely resembles the living tissue. Subsequently, the tissue-sample is dehydrated and embedded in an embedding media, predominantly paraffin wax, which allows the tissue to be cut in sections of as little as 2 μm.
The fixation however causes some loss of antigenicity. The modifications introduced in the proteins either chemically changes the epitopes recognized by the antibodies or physically prevent access to the epitopes. Many antigen retrieval or target retrieval methods have been developed to restore the antigenicity of the tissue, and most fall in one of two groups.
One traditional method comprises treating the tissue with a proteolytic enzyme, which opens up the tissue to allow the antibodies access to the antigens. For some specific antibodies, this method is the preferred antigen retrieval procedure, but it suffers from some limitations. Since the proteolytic enzymes degrade the tissue-proteins, there is a significant risk of destroying the specific epitope in the protein that is recognized by the antibody, thus the antigenicity may be lost rather than being restored. Furthermore, prolonged proteolytic treatment may cause the proteins to loose their three-dimensional structure, which is also significant for antibody-antigen recognition. These factors obviously necessitate close control with incubation times, however the required incubation time is very dependant on the activity of the enzyme.
During storage, the enzymes will slowly degrade themselves, particularly if the storage temperature is higher than recommended, and thus the activity of the enzyme-solution may vary greatly with time. This means that the optimal incubation time changes with the age of the enzyme-solution and ideally it should be adjusted accordingly.
Another factor that affects the efficiency of proteolytic target retrieval is the length of fixation of the tissue. The degree of cross-linking in the tissue increases with the fixation time and thus tissue that has been fixed for 72 hours will need longer proteolytic treatment than one that has been fixed for 24 hours. Frequently, however the information concerning fixation time is not available, in which case it is not possible to make the corresponding adjustment in enzyme incubation time.
A fundamentally different method comprises of boiling the tissue in an aqueous solution also known as heat induced antigen retrieval (HIAR). This pre-treatment method is not as dependent on fixation time as the enzymatic pre-treatment, and is more widely used. The high temperature accelerates hydrolysis of the aldehyde-induced modifications and the aldehyde released is diluted in the water, thus preventing it from reacting with the proteins again.
In conventional HIAR, typical boiling times are between 20 and 40 minutes at 95-99° C., dependent on the antigen, to which is added a cooling time of approximately 20 minutes before the slides can be removed safely without any risk of drying out the tissue. When the jar with the cold target retrieval solution is placed in a pre-heated water bath it will take 20 to 30 minutes before it reaches 95-99° C., which has to be added to the total procedure time. Using a microwave oven can reduce this heating step to 2-3 minutes as described in U.S. Pat. No. 5,244,787, (herein incorporated by reference) however the boiling and cooling times are unchanged, and thus the total procedure still amounts to 45 to 60 minutes. Heating the slides and target retrieval solution in a pressure cooker or similar pressurized device can reduce the boiling time. Here the tissue can be boiled at temperatures above 100° C. This is of particular importance at high altitudes, where the boiling point of aqueous solutions is lowered compared to sea level. In spite of the relatively short boiling time, the entire HIAR protocol is similar in length to the non-pressurised protocol, since cooling the liquid to a temperature where the pressure cooker can be opened safely is very time-consuming.
In addition to pure distilled water, many reagents are used routinely for antigen retrieval including Tris, Urea, EDTA, Citrate and saline buffers. Furthermore many solutions contain detergents, the selection comprising both ionic and non-ionic surfactants. In spite of the extensive research in the area of staining enhancement, no universal method or reagent has been identified that works with all subsequent immunohistochemical or immunocytochemical procedures.
Previous reports describe the use of glycerol for antigen retrieval purposes. Beebe (Beebe, K., Microscopy Today, 1999, Iss 9 (November), 30-31) has reported using 80% glycerol in a conventional submerging antigen retrieval method.
While traditional heat induced antigen retrieval restores tissue antigenicity for a wide variety of antibodies it has serious drawbacks when it comes to automation of the procedure. The use of near-boiling buffers in a tank is difficult to incorporate into an immunohistochemistry staining-instrument. Pumping buffer in and out of the tank and heating the buffer-solution is time-consuming and the energy-consumption associated with boiling the tissue is very high. Water-evaporation from the buffer-solution is high, and additional buffer must be added during the incubation-time. Furthermore, the steam can cause burns on the operators and it will condense in other parts of the instrument, where it may jeopardize the electronic circuits of the instrument.
Altogether, these processes significantly add to the complexity of a staining-instrument, and such factors obviously affect the expenses for the instrument.
Ventana Medical Systems have on their Benchmark® instrument a horizontal target retrieval procedure, where the aqueous buffer on the glass-slide is covered by an oil, the so called Liquid Coverslip™, to reduce evaporation during heating. Furthermore, the humidity inside the instrument is kept at near 100%.
Another example of an instrument that does antigen retrieval as well as staining is the Bond-Max® from Vision Biosystems. They use a cover-tile that is placed over the slide.
Both of the instruments mentioned above have the problem that any liquid will evaporate relatively quickly at a temperature near its boiling temperature and both instruments require the use of a cover over the sample.